DISTINCT SMAD1/SMAD5-MEDIATED FUNCTIONS IN TIP VERSUS STALK CELLS REGULATE SPROUTING ANGIOGENESIS

Blood vessel formation is critical for growth during development and postnatal life. Failure to establish a hierarchical branched blood vessel pattern leads to early embryonic lethality, and deregulation of the vessel patterning after birth contributes to the development of inflammatory, chronic and malignant diseases.Vascular patterning involves guided vessel sprouting, branching and fusion to form a primary network, and subsequent vessel network remodeling. These angiogenic sprouting processes are governed by orchestrated communication between cells in the tissue and the endothelial cells that line the blood vessels. The formation of new blood vessels is largely controlled by diffusible Vascular Endothelial Growth Factor (VEGF) that is produced by cells in hypoxic environments. The resulting gradient of VEGF will trigger in neighboring endothelium via binding to its receptor VEGFR2 - the selection of single endothelial cells (ECs) that will become the leading tip cells, or leader cells, of emerging sprouts. Tip cells are highly invasive and motile cells. Furthermore, they extend numerous filopodia to sense guidance cues in the surrounding microenvironment and to guide the sprout towards the source of VEGF in the hypoxic tissue. The high levels of VEGFR2 signaling experienced by tip cells result in enrichment of the Notch ligand Delta-like 4 (Dll4) in these leading cells. As a consequence of the increased levels of Dll4, they induce higher levels of Notch activity in adjacent ECs, and thus instruct the adjacent cells to acquire a stalk cell identity via Dll4/Notch-mediated lateral inhibition. The increased Notch activity in the stalk cells, or following cells, results in a transient decrease in responsiveness to the tip cell inducing stimuli because the targets of Notch signaling, Hes and Hey/Herp, encode transcriptional repressors that can directly down regulate the expression levels of VEGFR2, VEGFR3 and Dll4. Stalk cells are characterized by the production of higher levels of the target genes for Notch, VEGFR1 and Jagged1, while the production levels of VEGFR2/3 and Dll4 are higher in the tip cells. Genetic studies have demonstrated that Notch mediated lateral inhibition is crucial to balance the number of tip and stalk cells during sprouting angiogenesis.Many members of the BMP and TGFß signaling pathways play pivotal but often poorly defined roles in angiogenesis. Yet, their importance is highlighted by an increasing list of human vascular disorders related to perturbations in these signaling cascades. Furthermore, studies using different mouse models deficient in components of the BMP/TGFb signaling cascade have demonstrated that such animals develop severe cardiovascular defects. However, a complete understanding on the specific BMP/TGFb roles in angiogenesis is obscured by confounding observations in the field. In this project, we investigated the developmental defects in mouse embryos that lack two intracellular signal transducers of the BMP signaling cascade exclusively in endothelium, and studied some of the regulation mechanisms in further detail using a plethora of cell culture assays. We report that Smad1/Smad5 mediated BMP/TGFß signaling has different crucial functions in endothelium. I) It regulates directed cell migration of tip cells, and ii) synergism between Smad1/5 mediated BMP signaling via Id-proteins and Notch signaling is required for the efficient selection of tip and stalk cells. The endothelium-specific inactivation of Smad1/Smad5 in mouse embryos results in impaired Dll4/Notch signaling and increased numbers of tip cell-like cells at the expense of stalk cells. siRNA mediated Smad1/5 down regulation in human umbilical vein endothelial cells (HUVECs) impaired the expression levels of several target genes of Notch and of other stalk cell-enriched transcripts (Hes1, Hey1, Jagged1, VEGFR1 and Id1-3). Furthermore, Id proteins act as competence factors for stalk cell specification and form complexes with Hes1, which augment Hes1 levels in the endothelium. In contrast, pharmacological inhibition of Notch signaling induced ectopic expression of BMP responsive Id-genes. Our findings provide the first in vivo evidence for a regulatory loop between BMP/TGFß-Smad1/5 and Notch signaling that orchestrates tip versus stalk cell specification and vessel plasticity. In addition, we show that embryos with only one functional allele of either Smad1 or Smad5 in endothelium still die a little later during gestation (E14.5) due to severe cardiovascular and lymphatic defects. In these embryos, we describe that arterial ECs have a mixed identity and produce proteins associated with the arterial and lymphatic endothelium. These results put the BMP/TGFß signaling pathway forward as a novel regulator of EC plasticity and identity, regulation of directed cell migration, and stabilization of the number of tip/stalk cells during mouse early-onset sprouting angiogenesis.TABLE OF CONTENTS List of Figures xi List of Tables xiii List of Abbreviations xv Nederlandse Samenvatting xix Summary xxiii CHAPTER 1 – Introduction 1 1.1 Origin of ECs and vascular networks 2 1.1.1 Vasculogenesis 2 1.1.2 Angiogenesis 5 1.1.2.1 Sprouting angiogenesis 6 1.1.2.1.1 Tip and stalk selection 6 1.1.2.1.2 Changes in EC polarity, extracellular matrix degradation and migration 9 1.1.2.1.3 Anastomosis and lumen formation 11 1.1.2.2 Cell–cell adhesion and migration during sprouting angiogenesis 13 1.1.2.3 Intussusceptive angiogenesis 15 1.1.2.4 Arteriovenous differentiation 16 1.1.3 Lymphangiogenesis 21 1.1.3.1 Venous origin of the lymphatic system 22 1.2 BMP and TGFβ signaling cascades 25 1.2.1 BMP and TGFβ signaling in human vascular disease 27 1.2.1.1 Hereditary hemorrhagic telangiectasia 27 1.2.1.2 Pulmonary arterial hypertension 29 1.2.1.3 Tumor angiogenesis 29 1.2.2 BMP/TGFβ signaling in vascular development 30 1.2.2.1 Dual effects of BMP on angiogenesis 34 1.2.2.2 Dual effects of TGFβ on angiogenesis 36 1.2.2.3 Crosstalk between BMPs and Notch signaling cascades 37 1.2.2.4 Targeted inactivation of Smad5 results in angiogenesis defects 39 CHAPTER 2 – Objectives 41 CHAPTER 3 – Experimental procedures 43 3.1 Animals 43 VIII 3.2 Whole-mount detection of β-galactosidase activity, TUNEL assay 44 3.3 Immunofluorescence and antibodies 44 3.4 Culture of dorsal midgestation embryo explants 45 3.5 Cell culture procedures 46 3.5.1 Cell culture and transfection 46 3.5.2 Matrigel tube formation assay and time course experiments 46 3.5.3 Cell migration assays 47 3.5.4 In situ proximity ligation assay (PLA) 48 3.6 Quantifications 48 3.7 Gene expression analysis 49 3.8 Western blot analysis 50 3.9 Statistical evaluation 50 CHAPTER 4 – Results: Smad1 and Smad5 mediated regulation of angiogenesis and lymphangiogenesis 51 4.1 Gene dosage effect for Smad1 and Smad5 in the endothelium 52 4.2 Arteriovenous and lymphatic identity in Tie2Cre;(Smad1;Smad5)3fl/wt embryos 56 4.3 Discussion 59 CHAPTER 5 – Results: Smad1 and Smad5 are dispensable for vasculogenesis but crucial for angiogenesis 61 5.1 Smad1 and Smad5 mediated signaling is required for the developing vasculature 61 5.2 Smad1/5 signaling regulates sprouting angiogenesis 65 5.3 Pericyte recruitment and EC proliferation 68 5.4 Smad1/5-mediated signaling regulates tip cell polarity 70 5.5 Discussion 72 CHAPTER 6 – Results: Smad1/5-mediated signaling via Id proteins regulate stalk cell fate 75 6.1 Ubiquitous phosphorylation of Smad1 and Smad5 in the endothelium 75 6.2 Smad1 and Smad5 regulate stalk cell competence via Id proteins 77 6.3 Id-expressing ECs are excluded from the tip cell position 79 6.4 Discussion 82 IX CHAPTER 7 – Results: Synergism and antagonism between BMP and Notch signaling 85 7.1 Mutual interdependence of Notch and Smad1/5-mediated signaling 86 7.2 Smad1/5 deficiency impair Notch signaling in angiogenic endothelium 89 7.3 Smad1/5 deficiency results in loss of stalk cell marker expression 90 7.4 Discussion 93 CHAPTER 8 – Results: Dynamic Id protein-protein interactions with downstream targets of Notch 97 8.1 Id proteins stabilize Hes1 expression 98 8.2 Id proteins form complexes with Hes1 in ECs 99 8.3 Sequential interaction of Id proteins with Hes1 and Hey1 102 8.4 Discussion 104 CHAPTER 9 – General discussion 105 9.1 Model for the role of Smad1/Smad5 in the Dll4/Notch mediated stalk cell selection 106 9.2 Future perspectives 111 REFERENCES 117 PROFESSIONAL CAREER 129nrpages: 155status: publishe